This invention relates to novel methods and bacteria for the increased dinitrogen (N2) fixation of nodulating leguminous plants. More specifically, the present invention is directed to herbicide resistant N2 fixing bacteria, which have been isolated through selection of mutants or resistant variants or genetically engineered for herbicide resistance, and to nodulating leguminous plants infected with the herbicide resistant N2 fixing bacteria. The present invention is also directed to methods to enhance N2 fixation, nodulation, growth and/or grain yield of leguminous herbicide resistant or tolerant plants treated with the herbicide. The present invention is especially useful for increasing competitiveness of superior dinitrogen fixing strains over indigenous strains which are not resistant, under field conditions. Leguminous plants or seeds which are treated with a herbicide of interest are inoculated with the novel herbicide resistant rhizobia, preferably by applying the bacteria to the seeds or introducing the bacteria into the seed furrows at the time of planting, such that the novel rhizobia are able to form a symbiotic relationship with the plant.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Leguminous plants, such as soybean (Glycine max [L.] Merrill), form a symbiotic relationship with N2 fixing bacteria that allows the association of the two organisms to reduce (“fix”) dinitrogen gas in the atmosphere into a form which the plants can use as a nitrogen source. As a result, the plants do not require extensive nitrogen fertilization. The bacteria forming symbioses with legumes belong to the family Rhizobiaceae, which are a diverse group of gram-negative, nonspore-forming, rod-shaped, aerobic bacteria belonging to Group IV bacteria (Holt et al., 1994). Originally, rhizobia were classified as a single genus. More recent classifications, however, have divided rhizobia into distinct taxonomic groups based upon sequence similarities of 16s rRNA (Crawford et al., 2000). Current taxonomic divisions of rhizobia have grouped several clusters of rhizobia together within the alpha-proteobacteria based upon these sequence similarities. Among the most common genera within rhizobia are: Rhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium, and Bradyrhizobium. The ability of a rhizobia to nodulate its host exhibits specificity. For example, rhizobia which form symbiotic, N2 fixing nodules generally belong to one of many strains within rhizobia, including B. japonicum subspecies 1 and 2, B. elkanii and S. fredii (Devine and Kuykendall, 1996). The B. elkanii strains share a DNA homology and generally nodulate cowpea (Vigna unguiculata) as well as soybean. The B. elkanii strains can cause rhizobitoxine-induced chlorosis (Erdman et al., 1957). Some soybean plants can also enter symbiotic relationships with a fast-growing rhizobia species, Sinorhizobium fredii. 
The bacteria infect the root, forming a nodule where biological N2 fixation occurs that supplies 40 to 85% of the soybean's nitrogen requirements (Graham, 1998). Nitrogen fixation commences about three weeks after the infection process begins and is indicated by large, irregularly shaped nodules having a beef-steak red interior color due to the presence of leghemoglobin. Early in the growing season, nodules are clustered near the root crown. Later, the nodules located on secondary roots become more important in N2 fixation activity. Nodules must be present for N2 fixation to occur. However, not all nodules are effective. Effectiveness of nodules is reflected in the ability of the nodules to fix dinitrogen. Effective nodules are those nodules formed on legume roots that have the ability to fix (reduce) N2 symbiotically at high rates relative to a recognized superior rhizobial strain which serves as a standard, such as strain USDA 110 for B. japonicum infecting soybean. Rates of N2 fixation may be determined directly by quantifying the amount of 15N-labeled organic nitrogen per unit time per unit nodule mass following exposure of nodules to 15N2 for a set period of time. Alternatively, effective nodules may be determined indirectly by quantifying the conversion rate of acetylene to ethylene by nodules formed by a specific rhizobial strain relative to that of a recognized superior rhizobial strain which serves as a standard, such as strain USDA 110 for B. japonicum. The rate of acetylene reduction is measured by standard methods (Purcell et al., 1997; King et al., 2001) of flowing 1:9 acetylene:air mixture at a constant and known flow rate through a sealed pot having an inlet and exit port and containing the nodulated roots of said legume. Gas samples are taken from the effluent stream with a syringe when the quotient of the pot volume to volumetric flow rate is four. Gas samples are injected into a gas chromatograph with a flame ionization detector for ethylene determination. Various screening studies indicate that, of the nodules on a typical soybean plant, approximately 25% are highly effective, 50% are of medium effectiveness, and 25% are ineffective. Thus, the number of nodules on the soybean root is not the only indicator of adequate N2 fixing ability.
Glyphosate [N-(phosphonomethyl)glycine] is the active ingredient in the non-selective herbicide Roundup™ (Monsanto Co., St. Louis, Mo. 63167). Advances in biotechnology have resulted in glyphosate resistant or tolerant (GR) soybean cultivars, providing an effective broad-spectrum postemergence weed-control option. Glyphosate competitively inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (E.C. 2.5.1.19), an enzyme in the shikimate pathway leading to the synthesis of aromatic amino acids (Duke, 1988). Commercial glyphosate resistant or tolerant (GR) soybean expresses an EPSPS that is resistant or tolerant to glyphosate. The gene for resistance was originally isolated from Agrobacterium sp. (Padgette et al., 1995). Extensive research on the effects of glyphosate on soybean in relatively high yield environments (2000 to 4000 kg ha−1) has indicated that there is no effect of glyphosate relative to untreated plants under weed free conditions (Delannay et al., 1995).
Glyphosate is not readily degraded in soybean and concentrates in metabolic sinks, such as young roots and developing and mature nodules (Duke, 1988). Previous research indicates that a single foliar application of glyphosate at 0.5 kg ha−1 can result in concentrations up to 0.3 mM in bulk root tissue of susceptible plant species (Honegger et al., 1986). Higher glyphosate use rates or repeated applications could result in even greater concentrations, especially in the stronger metabolic sinks such as soybean root nodules as compared to the bulk root system (McWhorter et al., 1980).
Symbiotic N2 fixation is critical for obtaining high yields in soybean grown on soils without large amounts of available nitrogen (Cooper and Jeffers, 1984). Importantly, N2 fixation in soybean is more sensitive to water-deficit stress than are other processes such as gas exchange (Durand et al., 1987), transpiration (Sall and Sinclair, 1991), and uptake and assimilation of inorganic soil nitrogen (Purcell and King, 1996).
Although EPSPS in GR soybean is resistant or tolerant to glyphosate, strains of the N2 fixing symbiont such as Bradyrhizobium japonicum have a sensitive form of the enzyme (Jaworski, 1972; Moorman et al., 1992). The sensitivity of B. japonicum to glyphosate is influenced by herbicide concentration and bacterial strain. Growth of B. japonicum strain, USDA 110, in culture has been inhibited 41 to 100% at glyphosate concentrations of 0.5 to 5 mM (Moorman et al., 1992). Strains USDA 123 and 138 are less sensitive at 0.5 to 1 mM glyphosate, with inhibition of only 10 to 20%, however, they are inhibited 100% at a 5 mM concentration. B. japonicum strain USDA 71 is very sensitive, with bacterial growth decreased 69 to 92% by glyphosate concentrations of 0.01 to 1 mM (Jaworski, 1972). Despite recognition of B. japonicum sensitivity to glyphosate, there have been no previous reports of the effect of glyphosate on N2 fixation, nodulation or biomass in GR soybean in symbiosis with rhizobia species.
Superior dinitrogen fixing strains have been selected and used to infect leguminous plants in an attempt to increase N2 fixation. While these strains have demonstrated increased N2 fixation in controlled environments, they have not been effective in field environments where the indigenous bacterial populations have out-competed them for sites on legume roots. Thus, there is a definite need for a method to increase competitiveness of superior nitrogen fixing strain against indigenous populations. The present invention solves this need as illustrated herein.